Pyrophosphate-dependent phosphofructokinase from the amoeba Naegleria fowleri, an AMP-sensitive enzyme

Abstract

PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.

Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila

“…The spirochete PP i -phosphofructokinase was virtually unaffected by traditional allosteric effectors and displayed hyperbolic kinetics for both the forward and reverse directions and is thus not likely to be allosterically regulated. This lack of apparent regulation is similar to what has been found with other bacterial and most primitive eukaryotic PP i -phosphofructokinases, except those from Naegleria fowleri and Euglena gracilis, which have been shown to be either activated by AMP or fructose 2,6-diphosphate, respectively (Enomoto et al 1988;Mertens et al 1993). Reaction rates in vivo in S. thermophila are thus likely to be in near-equilibrium, controlled simply by the level of enzyme activity and the concentration of the intracellular reactants and products.…”

“…The reverse reaction was conducted (50°C) using phosphoglucoisomerase and glucose-6-phosphate dehydrogenase as described by Mertens et al (1993) except the phosphoglucoisomerase concentration was doubled. Kinetic parameters for the reverse reaction were determined by varying the concentration of fructose 1,6-diphosphate or inorganic phosphate (P i ) with saturating quantities of either P i (10 mM) or fructose 1,6-diphosphate (5 mM), respectively.…”

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

“…The few PFKs characterized from protists, whether PP i -linked (Reeves 1968;Mertens et al 1989Mertens et al , 1993Mertens 1990;Peng and Mansour 1992;Denton et al 1994) or ATP-linked (Cronin and Tipton 1985), have subunit sizes ranging from 42 to 67 kDa. Although larger than the 35-kDa eubacterial subunit, this size still can accommodate only one substrate binding site and one phosphoryl-donor binding site and thus corresponds to the simplest types of PFKs known.…”

Comparative Metabolism of Free‐living Bodo saltans and Parasitic Trypanosomatids

“…), while the other is related to the PP i ‐PFK of Naegleria fowleri (Mertens et al. ), hence an ortholog of the bacterial and plant PFKs. Phylogenetic analysis (Fig.…”

“…The use of a PP i ‐PFK ensues an increased ATP yield during anaerobic glycolysis (Mertens et al. ). Thus, B. saltans may survive equally well in both high and low oxygen environments, while trypanosomatids, which have lost the PP i ‐dependent enzyme, are considered predominantly aerobic.…”